HMGI proteins in cancer

ABSTRACT

The present invention pertains to a method for detecting HMGI-C or HMGI(Y) as a diagnostic marker for benign or malignant tumors using a probe for a sample from a patient that recognizes HMGI-C or HMGI(Y). The method comprises the steps of (a) contacting HMGI-C or HMGI(Y) from a sample from a patient with a probe which binds to HMGI-C or HMGI(Y); and (b) analyzing for HMGI-C or HMGI(Y) by detecting levels of the probe bound to the HMGI-C or HMGI(Y). The presence of HMGI-C or HMGI(Y) in the sample is positive for a benign or malignant tumor. The present invention also pertains to a method for detecting antibodies to HMGI-C or HMGI(Y) as a diagnostic marker for benign or malignant tumors. The present invention further pertains to a method for treating benign and malignant tumors in a patient by blocking the biological activity of HMGI-C or HMGI(Y). The present invention also pertains to a method for treating obesity in a patient by blocking the biological activity of HMGI-C or HMGI(Y).

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during development of this invention utilizedUnited States Government funds. The United States Government has certainrights in this invention: NIH grant no. GM38731, HD30498, and1K11CA01498.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to a method for detecting HMGI-C orHMGI(Y) as a diagnostic marker for benign or malignant tumors using aprobe for a sample from a patient that recognizes HMGI-C or HMGI(Y). Themethod comprises the steps of (a) contacting HMGI-C or HMGI(Y) from asample from a patient with a probe which binds to HMGI-C or HMGI(Y); and(b) analyzing for HMGI-C or HMGI(Y) by detecting levels of the probebound to the HMGI-C or HMGI(Y). The presence of HMGI-C or HMGI(Y) in thesample is positive for a benign or malignant tumor. The presentinvention also pertains to a method for detecting antibodies to HMGI-Cor HMGI(Y) as a diagnostic marker for benign or malignant tumors. Thepresent invention further pertains to a method for treating benign andmalignant tumors in a patient by blocking the biological activity ofHMGI-C or HMGI(Y). The present invention also pertains to a method fortreating obesity in a patient by blocking the biological activity ofHMGI-C or HMGI(Y).

2. Description of the Background

The disclosures referred to herein to illustrate the background of theinvention and to provide additional detail with respect to its practiceare incorporated herein by reference and, for convenience, arereferenced in the following text and respectively grouped in theappended bibliography.

HMGI Proteins in Adipogenesis and Mesenchyme DifferentiationUnderstanding various genes and pathways underlying development ofmulticellular organisms provide insights into the molecular basis of thehighly regulated processes of cellular proliferation anddifferentiation. In turn, genetic aberrations in control of cell growthlead to a variety of developmental abnormalities and, most prominently,cancer (Aaronson, 1991). To pursue identification of genes involved inthese fundamental biological processes, the viable pygmy mutation(MacArthur, 1944) was investigated because it gives rise to mice ofsmall stature due to a disruption in overall growth and development ofthe mouse. An insertional transgenic mutant facilitated cloning of thelocus (Xiang et al., 1990) and subsequently it was shown that expressionof the HMGI-C gene was abrogated in three pygmy alleles (unpublishedresults).

HMGI-C belongs to the HMG (high mobility group) family of DNA-bindingproteins which are abundant, heterogeneous, non-histone components ofchromatin (Grosschedl et al., 1994). HMG proteins are divided into threedistinct families, the HMG box-containing HMG1/2, the active chromatinassociated HMG14/17 and the HMGI proteins (Grosschedl et al., 1994). Atpresent, the last family consists of two genes, HMGI(Y) (Johnson et al.,1988; Friedmann et al., 1993) which produces two proteins viaalternative splicing (Johnson et al., 1989) and HMGI-C (Manfioletti etal., 1991; Patel et al., 1994). A prominent feature of HMGI proteins isthe presence of DNA-binding domains which bind to the narrow minorgroove of A-T rich DNA (Reeves and Nissen, 1990) and are thereforereferred to as A-T hooks. Recently, valuable insights have been gainedinto their mechanism and role in transcription (Thanos and Maniatis,1992; Du et al., 1993). The HMGI proteins have no transcriptionalactivity per se (Wolffe, 1994), but through protein-protein andprotein-DNA interactions organize the framework of the nucleoprotein-DNAtranscriptional complex. This framework is attained by their ability tochange the conformation of DNA and these proteins are therefore termedarchitectural factors (Wolffe, 1994). In the well-studied case ofHMGI(Y) and the interferon B promoter, HMGI(Y) stimulates binding ofNF-KB and ATF-2 to appropriate sequences and alters the DNA structurewhich allows the two factors to interact with each other and presumablywith the basal transcription machinery (Thanos and Maniatis, 1992; Du etal., 1993).

A number of studies have revealed an association between increasedexpression levels of HMGI proteins and transformation (Giancotti et al.,1987, 1989, 1993). For example, in chemically, virally or spontaneouslyderived tumors, appreciable expression of HMGI-C was found in contrastto no detectable expression in normal tissues or untransformed cells(Giancotti et al., 1989). A recent study has demonstrated a more directrole for HMGI-C in transformation (Berlingieri et al., 1995). Cellsinfected with oncogenic retroviruses failed to exhibit variousphenotypic markers of transformation if HMGI-C protein synthesis wasspecifically inhibited.

DNA probes adjacent to HMGI-C were mapped to the distal portion of mousechromosome 10 in a region syntenic to the long arm of human chromosome12 including and distal to band q13 (Justice et al., 1990). This genomicregion is under intensive investigation because it is the location ofconsistent rearrangements in a number of neoplasms, mainly ofmesenchymal origin (Schoenberg Fejzo et al., 1995). Lipomas, tumorsmainly composed of mature fat cells, are one of the most commonmesenchymal neoplasms that occur in humans (Sreekantaiah et al., 1991).Approximately 50% of lipomas are characterized by cytogeneticrearrangements and the predominant alteration is a presumably balancedtranslocation involving 12q14-15 with a large variety of chromosomalpartners including 1,2,3,4,5,6,7,10,11,13,15,17,21, and X (Sreekantaiahet al., 1991; Fletcher et al., 1993). This variability in reciprocaltranslocations along with duplications, inversions, and deletions of12q14-15 in these tumors, strongly indicates a primary role of a gene onchromosome 12 in lipomas. Furthermore, this gene may play a key role innormal differentiation of primitive mesenchyme as not only lipomas, butalso uterine leiomyomas (smooth muscle tumors), lipoleiomyomas (smoothmuscle and adipose components), and pulmonary chondroid hamartomas(primitive mesenchyme, smooth muscle, adipose, and mature cartilagecomponents) are all clonal proliferations that are characterized byrearrangements of 12q14-15 (Schoenberg Fejzo et al., 1995).

Interestingly, breakpoints in a lipoma, a pulmonary chondroid hamartomaand uterine leiomyomata have been shown to map within a single YAC(Schoenberg Fejzo et al., 1995).

HMGI Proteins in Mammalian Growth and Development

The first step in the molecular definition of the pygmy mutation wasmade possible by the isolation of a transgenic insertional mouse mutantat the locus, pg^(TgN40ACha) (Xiang et al., 1990). A 0.5 kb ApaI—ApaIsingle copy genomic sequence 2 kb from the site of transgene insertionwas identified (Xiang et al., 1990) and used to initiate abi-directional chromosome walk on normal mouse genomic DNA. The analysisof seven overlapping clones spanning 91kb delineated a 56 kb commondeletion between two informative mutants, pg and pg^(TgN40Cha) (FIG. 8a).

The common area of disruption was investigated further for candidatetranscription units. The technique of exon amplification (Buckler etal., 1991) was employed to identify putative exons and clones 803 and5B, in the same orientation, produced spliced products (FIG. 8 b). Theirsequence was determined (Ausubel et al., 1988) and a comparison to DNAsequence databases (GenBank and EMBL) revealed 100% homology to apreviously identified gene, HMGI-C (Manfioletti et al., 1991) (FIG. 8c). The HMGI members have been assigned multiple functions (Manfiolettiet al., 1991) and recently, have been shown to play a critical role inregulation of gene expression as architectural factors by inducing DNAconformational changes in the formation of the three-dimensionaltranscription complex (Thanos & Maniatis, 1992; Du, W. et al., 1993).

Subsequently, the genomic structure of HMGI-C revealed that the genecontains five exons and spans a region of approximately 110 kb (FIG. 8d).

Single copy sequences from the 190 kb cloned pygmy locus, surroundingand including the HMGI-C gene (FIG. 8 d), were used as probes onSouthern blots containing DNA isolated from the two informative alleles(Xiang et al., 1990).

The genomic area encompassing HMGI-C is completely deleted in thetransgenic insertional mutant pg^(TgN40ACha) (A/A), whereas in thespontaneous mutant pg, the 5′ sequences and the first two exons areabsent (FIG. 8 d).

Misexpression of Disrupted HMGI Proteins in Human Tumors

Cancer arises from aberrations in the genetic mechanisms that controlgrowth and differentiation and ongoing elucidation of these mechanismscontinues to improve the understanding of mammalian development and itsvarious abnormalities. Increasingly, accumulating experimental evidencepoints towards transcriptional deregulation as one of the pivotal eventsin neoplasia. Many of the known transforming retroviral oncogenes, suchas v-myc, v-fos and v-myb, are homologs of mammalian transcriptionfactors which are normally involved in proliferation and differentiationcontrol. Genes that encode for such transcription factors are frequentlyaffected by the somatically acquired genetic changes which arisestochastically over a lifetime of an organism. These alterations, whichcan either activate expression of the relevant genes or disrupt them tocreate novel fusion proteins, affect transcription networks and initiatecancer.

One of the transcription factors whose disruption was shown to result intumorigenesis is HMGI-C, which has attracted considerable attention fortwo reasons. First, a series of elegant experiments demonstrated thatHMGI(Y) is involved in transcriptional regulation and is required forvirus induction of the human interferon-β gene expression. Theseobservations were incorporated into a novel model in which activation ofgene expression is initiated by a higher order transcription enhancercomplex. This functional nucleoprotein entity termed enhanceosome isformed when several distinct transcription factors assemble on DNA in astereospecific manner. Combinatorial mechanisms of the enhanceosomeformation enable the cell to achieve high specificity of gene activationin response to multiple biological stimuli. As an essential component ofthe enhanceosome, HMGI(Y) promotes the assembly of thisthree-dimensional structure through both protein-protein and protein-DNAinteractions. The latter activity is mediated through the HMGIDNA-binding domains.

The function of HMGI-C, the other known member of the HMGI family, ingrowth and development control is better understood at the biologicallevel. In humans, rearrangements of HMGI-C were linked to thepathogenesis of several distinct types of solid tumors. Rearrangementsof the chromosomal band 12q13-15, consistently found in a wide varietyof benign mesenchymal neoplasms, disrupt HMGI-C and generate novelchimeric transcripts. In the vast majority of the analyzed tumors, thesetranscripts consist of the HMGI-C DNA-binding domains fused to ectopicsequences provided by the translocation partner.

In the mouse, HMGI-C inactivation produced a dramatic disruption of bothpre- and postnatal growth, resulting in the pygmy phenotype. Pygmy miceexhibit significant growth retardation which is first apparent inmidgestation and becomes even more pronounced after birth. Adult animalsare proportionally built and viable but exhibit a 60% weight reductioncompared to their wildtype littermates. A detailed phenotypic analysisof the pygmy mouse revealed that the weight reduction in most of thetissues is commensurate with the overall decrease in body weight. Mostinterestingly, HMGI-C inactivation does not affect the growthhormone-insuline-like growth factor endocrine pathway, suggesting thatHMGI-C functions in a previously unknown growth regulatory mechanism.

The molecular basis of the pygmy mutation is not well understood. Highlevels of the HMGI proteins are not required for cell growth per se andelevated HMGI expression appear to be associated with the biologicalstate of the cell more directly than with its high proliferation rate.Upon transformation with oncogenic retroviruses, expression of HMGI-Cand HMGI(Y) in epithelial cells is dramatically increased even thoughthe proliferative capacity of the infected cells remains unaffected.Furthermore, analysis of a transformed cell line which retained itsdifferentiated phenotype revealed that levels of the HMGI expressionwere significantly lower than in cell lines which lost theirdifferentiation markers as a result of transformation. Other studiesdemonstrated that HMGI-C is expressed in less differentiated mesenchymalcells but is no longer present in their terminally differentiatedcounterparts. In combination, these results indicate that the functionof the HMGI proteins may be to maintain the undifferentiated cellularstate.

The diverse set of mesenchymal neoplasms in which HMGI-C is frequentlydisrupted by translocations of 12q13-15 includes lipomas, uterineleiomyoma, pulmonary hamartoma and pleomorphic adenomas of salivarygland. Another cytogenetic subgroup which can be identified in this setof tumors is characterized by rearrangements at 6p21-23. Intriguingly,HMGI(Y) has previously been localized to this chromosomal area.

Translocation Breakpoints Upstream of the HMGI-C Gene in UterineLeiomyomata

Uterine leiomyomata, also known as fibroids, are the most common pelvictumors in women. Systematic histologic examination of hysterectomyspecimens has shown a prevalence as high as 77% for these tumors inwomen of reproductive age. Although benign, uterine leiomyomataconstitute a major health problem as they are associated with abnormaluterine bleeding, pelvic pain, urinary incontinence, spontaneousabortion, premature delivery, and infertility. Symptomatic fibroids arethe leading indication for hysterectomy, accounting for 27% of theestimated 680,000 procedures performed annually in the United States.

Several different consistent chromosomal rearrangements have beenidentified in uterine leiomyomata, and they suggest involvement of acritical gene on chromosome 12 in the pathobiology. A translocationinvolving chromosomes 12 and 14, t(12;14)(q14-15;q23-24), represents oneof the most common rearrangements, although trisomy 12, inversions andduplications of 12q14-q15, and translocations of 12q14-q15 withchromosomes other than 14 are not uncommon. The breakpoint in 12q14-q15in uterine leiomyomata is in an intriguing chromosomal region because itis also the location of consistent rearrangements in other benign solidtumors, including lipomas and pleomorphic adenomas of the salivarygland. Rearrangements of 12q13-15 have been reported in pulmonarychondroid hamartoma, endometrial polyps, epithelial breast tumors,hemangiopericytoma, and an aggressive angiomyxoma. These tumors have thecommon properties of being mesenchyme-derived and benign. Therefore, ithas been hypothesized that a single gene involved in mesenchymedifferentiation and growth could be responsible for these multiple tumortypes.

H. R. Asher et al. (1995) reported that HMGI-C, an architectural factorthat functions in transcriptional regulation, is disrupted byrearrangement at 12q14-15 chromosomal breakpoint in lipomas and suggestsa role for HMGI-C in adipogenesis and mesenchyme differentiation.

X. Zhou et al., (1995) shows that the pygmy phenotype arises from theinactivation of HMGI-C which function as architectural factors in thenuclear scaffold and are critical in the assembly of stereospecifictranscriptional complexes.

SUMMARY OF THE INVENTION

The present invention pertains to a method for detecting HMGI-C orHMGI(Y) as a diagnostic marker for benign or malignant tumors using aprobe for a sample, or an extract thereof, from a patient thatrecognizes HMGI-C or HMGI(Y), which comprises the steps of:

(a) contacting HMGI-C or HMGI(Y) from a sample from a patient with aprobe which binds to HMGI-C or HMGI(Y); and

(b) analyzing for HMGI-C or HMGI(Y) by detecting levels of the probebound to the HMGI-C or HMGI(Y), wherein the presence of HMGI-C orHMGI(Y) in the sample is positive for a benign or malignant tumor.

The present invention also pertains to a method for detecting antibodiesto HMGI-C or HMGI(Y) as a diagnostic marker for benign or malignanttumors using a probe for a sample, or an extract thereof, from a patientthat recognizes antibodies to HMGI-C or HMGI(Y), which comprises thesteps of:

(a) contacting antibodies to HMGI-C or HMGI(Y) from a sample from apatient with a probe which binds to antibodies to HMGI-C or HMGI(Y); and

(b) analyzing for antibodies to HMGI-C or HMGI(Y) by detecting levels ofthe probe bound to the antibodies to HMGI-C or HMGI(Y), wherein thepresence of antibodies to HMGI-C or HMGI(Y) in the sample is positivefor a benign or malignant tumor.

The present invention further pertains to a method for treating benignand malignant tumors in a patient by blocking the biological activity ofHMGI-C or HMGI(Y) which comprises adminstering to the patient atherapeutically effective amount of an inhibitor specific for HMGI-C orHMGI(Y).

The present invention still further pertains to a method for treatingobesity in a patient by blocking the biological activity of HMGI-C orHMGI(Y) which comprises adminstering to the patient a therapeuticallyeffective amount of an inhibitor specific for HMGI-C or HMGI(Y).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(A) and 1(B) illustrate the genomic structure of the human HMGI-Cgene.

FIGS. 2(A) through 2(F) illustrate FISH mapping of HMGI-C lambda clonesto lipoma tumor metaphase chromosomes from three lipomas revealingrearrangement of HMGI-C in all three tumors.

FIG. 3 illustrates RT-PCR amplification of HMGI-C chimeric transcripts.

FIGS. 4(a) through 4(c) illustrates rearrangements of 12q15 in humanlipomas which disrupt the HMGI-C gene and produce chimeric transcripts.

FIG. 5 illustrates RT-PCR using primers located on either side of thefusion site between HMGI-C and novel sequences.

FIGS. 6(A) and 6(B) illustrate novel sequences fused to the DNAbinding-domains of HMGI-C which encode transcriptional regulatorydomains.

FIGS. 7(a) through 7(c) illustrates the structure and domainorganization of HMGI-C and the predicted fusion proteins.

FIGS. 8(A) through (D) illustrate the identification and genomiccharacterization of the HMGI-C gene at the pygmy locus in normal andmutant alleles.

FIG. 9 illustrates HMGI-C gene expression of three alleles at the mousepygmy locus.

FIGS. 10(A) through (C) illustrate targeted disruption of the HMGI-Cgene.

FIGS. 11(A) through (C) illustrate expression of HMGI-C in developmentand growth.

DETAILED DESCRIPTION OF THE INVENTION

Aberrations in the genetic mechanisms that control growth andproliferation have emerged as a primary event in carcinogenesis. Thefunction of HMGI-C and HMGI(Y), two embryonically expressed DNA-bindingproteins, was investigated because their expression is highly associatedwith tumor development.

Disruptions of either HMGI-C or HMGI(Y) in humans result in a diversearray of solid mesenchymal tumors. Most prominent among these neoplasmsare uterine leiomyomata, the most common pelvic tumors in women and theindication for over 200,000 hysterectomies annually in the UnitedStates. In tumors of mammary and thyroid glands as well as in prostatecancer, HMGI expression is highly correlated with tumor progression andmetastasis, suggesting that these proteins can be used for asprogression markers for a variety of tumor types.

Further proof for the pivotal role of HMGI proteins in both normal andpathological growth was obtained in the mouse system. Homologousrecombination was used to inactivate murine HMGI-C gene. Demonstratingthe importance of the HMGI genes in growth regulation, HMGI-C knockoutmice exhibit significant growth retardation (mutant mice are 60% smallerthan their wild-type littermates) with the reduction in most tissuescommensurate with the overall decrease in the body weight. Even moreimportantly, these pygmy mice are highly resistant to chemically inducedskin cancer. Specifically, the frequency of tumor development in theknockout mice is 40% of that in the control animals and tumormultiplicity exhibits a 20-fold decrease. Independently, inhibition ofHMGI-C synthesis was shown to render thyroid epithelial cellsintransigent to retroviral transformation. At the molecular level, HMGIproteins function in transcriptional regulation by promoting cooperativebinding of the transcription factors to DNA. Deregulation of thedownstream target genes can easily account for the important biologicalroles of the HMGI proteins as well as for the dramatic consequences oftheir inappropriate expression.

Lipomas are one of the most common mesenchymal neoplasms in humans. Theyare characterized by consistent cytogenetic aberrations involvingchromosome 12 in bands q14-15. Interestingly, this region is also thesite of rearrangement for other mesenchymally derived tumors. Thepresent invention demonstrates that HMGI-C, an architectural factor thatfunctions in transcriptional regulation, has been disrupted byrearrangement at the 12q14-15 chromosomal breakpoint in lipomas.Chimeric transcripts were isolated from two lipomas in which HMGI-CDNA-binding domains (A-T hook motifs) are fused to either a LIM or anacidic transactivation domain. These results identify the first generearranged in a benign neoplastic process that does not proceed to amalignancy and suggest a role for HMGI-C in adipogenesis and mesenchymedifferentiation.

HMGI-C is an attractive candidate gene to be implicated in lipomaformation. This gene is required in transformation (Berlingieri et al.,1995) and is a transcriptional regulatory factor as are many genesidentified at translocation breakpoints in a variety of tumors(Rabbitts, 1994). Secondly, disruption of HMGI-C leads to mice of smallstature which, most intriguingly, have disproportionately less body fatthan normal littermates (Benson and Chada, 1994). Finally, mouse HMGI-Cmaps to a region syntenic to human 12q14-15 which is the area mostfrequently rearranged in lipomas (Mandahl et al., 1988). Therefore, thehuman homolog of the mouse HMGI-C gene was cloned and its possible rolein lipomas investigated.

Growth is one of the fundamental aspects in the development of anorganism. Classical genetic studies have isolated four viable,spontaneous mouse mutants (Green, 1989) disrupted in growth, leading todwarfism. Pygmy is unique among these mutants because its phenotypecannot be explained by aberrations in the growth hormone-insulin-likegrowth factor endocrine pathway (Lin, 1993; Li, et al., 1990; Sinha etal., 1979; Nissley et al., 1980). The present invention shows that thepygmy phenotype arises from the inactivation of HMGI-C and are criticalin the assembly of stereospecific transcriptional complexes (Tjian &Maniatis, 1994). In addition, HMGI-C and the other HMGI family member,HMGI(Y)(Johnson et al., 1988), were found to be expressed predominantlyduring embryogenesis. The HMGI family are known to be regulated by cellcycle dependent phosphorylation which alters their DNA binding affinity(Reeves et al., 1991). Overall, these results demonstrate the importantrole of HMGI proteins in mammalian growth and development.

Among the most prominent characteristics consistently exhibited bycancer cells are karyotypic aberrations which disturb genes essentialfor the regulation of fundamental cellular processes. A wide array ofsolid mesenchymal tumors is characterized by recurrent rearrangements ofchromosomal bands 12q13-15 or 6p21-23. This study shows that HMGIexpression is normally restricted to undifferentiated, rapidly dividingcells but is activated in differentiated adipocytes followingtranslocations of 12q13-15 or 6p21-23 in human lipomas. The presentinvention shows that the molecular pathway of tumor development isdictated by the precise nature of HMGI disruption and that HMGImisexpression in a differentiated cell is a pivotal event in benigntumorigenesis.

Uterine leiomyomata are the most common pelvic tumors in women and arethe indication for more than 200,000 hysterectomies annually in theUnited States. Rearrangement of chromosome 12 in bands q14-q15 ischaracteristic of uterine leiomyomata and other benign mesenchymaltumors, and a YAC spanning chromosome 12 translocation breakpoints wasidentified in a uterine leiomyoma, pulmonary chondroid hamartoma, andlipoma. Recently, it was demonstrated that HMGI-C, an architecturalfactor mapping within the YAC, is disrupted in lipomas, resulting innovel fusion transcripts. This study concerns the localization oftranslocation breakpoints in seven uterine leiomyomata 10 to >100 kbupstream of HMGI-C by use of fluorescence in situ hybridization. Thesefindings suggest a different pathobiologic mechanism in uterineleiomyomata from that in lipomas. HMGI-C is the first gene identified inchromosomal rearrangements in uterine leiomyomata and has importantimplications for an understanding of benign mesenchymal proliferationand differentiation.

Recently, molecular dissection of this chromosomal region hassubstantiated this hypothesis. To identify a gene at the breakpoint onchromosome 12 in uterine leiomyomata, a high-density physical map of thet(12;14) breakpoint region was constructed and identified a YAC, 981f11,that spans the translocation breakpoints in a uterine leiomyomata,pulmonary chondroid hamartoma and a lipoma. Further detailedcharacterization showed that the gene for HMGI-C, an architecturalfactor that is a non-histone component of chromatin, maps within 981f11and is disrupted in lipomas. HMGI-C is rearranged in lipomas withchromosome 12 translocations, resulting in novel chimeric transcriptsthat fuse the DNA-binding A-T hook domains of HMGIC with potentialtranscriptional activation domains.

Furthermore, there is evidence that HMGI-C plays a role in mesenchymalcell proliferation.

RESULTS HMGI Proteins in Adipogenesis and Mesenchyme Differentiation

Genomic Isolation and Characterization of the Human HMGI-C Gene

To obtain genomic clones of HMGI-C, DNA from yeast strains harboringYACs, yWPR383 and yWPR384 were subcloned into the lambda FIXII vector.Because there is extensive conservation (96%) between mouse and humanHMGI-C homologs (Patel et al., 1994), mouse HMGI-C cDNA fragmentsencompassing all five exons were used as probes on lambda libraries andfive clones were isolated (FIG. 1A). Restriction mapping of lambdaclones followed by Southern blot analysis allowed identification ofvarious restriction fragments containing cross-hybridizing sequences.These fragments were subcloned and nucleotide sequence analysisconfirmed published data (Patel et al., 1994). The first three exonseach contain a DNA binding domain encoding the A-T hook motif that ischaracteristic of the HMGI family (Reeves and Nissen, 1990) and exons 4and 5 encode the acidic domain of the molecule (Manfioletti et al.,1991) (FIG. 1B). Notably, a large intron (>25 kb) between exons 3 and 4separates the DNA binding domains from the remainder of the protein(FIG. 1B).

Fluorescence In Situ Hybridization of Lambda HMGI-C Exon Clones toLipoma Metaphase Chromosomes

Lambda clones from 5′ and 3′ ends of HMGI-C were used as probes for FISHto tumor metaphase chromosomes. In lipoma ST90-375 containing at(12;15)(q15;q24) translocation, lambda clone H403 which contains the 5′end of the gene gave a hybridization signal on the der(12), thus mappingproximal to the breakpoint. In contrast, lambda clone H4002 whichcontains a portion of the 3′ end of the HMGI-C gene, gave ahybridization signal on the der(15) and therefore maps distal to thebreakpoint (FIG. 2). This result is consistent with a disruption ofHMGI-C due to the t(12; 15) in this lipoma. Two other lipomas withtranslocations in 12q15 were studied, similarly. In ST93-724 containinga t(3;12)(q29;q15), lambda clone H409 containing the 5′ end of HMGI-Chybridized to the der(12), while the 3′ end clone H4002 hybridized tothe der(3) (FIG. 2). In ST91∝198 with a t(12;13)(q14-22;q21-32), the 5′clone H403 mapped on the der(13) suggesting a position distal to thebreakpoint. However, from the 3′ end, no hybridization to eitherderivative chromosome was noted in 20/20 metaphases using lambda cloneH4002 indicating that this portion of HMGI-C is deleted (FIG. 2).Therefore, in this tumor, the translocation appears to be proximal toHMGI-C with the 5′ end of the gene retained but the 3′ end deleted.Regardless of the chromosomal mechanism which may include a complexrearrangment in ST91198, HMGI-C is disrupted in three out of threelipomas analyzed.

Identification of Chimeric Transcripts

The molecular structure of the HMGI-C transcripts in the lipomas wasnext investigated. Total mRNA was isolated (Chirgwin et al., 1979) fromprimary cell cultures of ST90-375 t(12;15) and ST93-724 t(3;12) and 3′RACE performed (Frohman et al., 1988). The resulting products wereanalyzed by agarose gel electrophoresis and DNA fragments of size 441and 627 bp were obtained from RNA samples isolated from ST90-375 andST93-724, respectively (FIG. 3). These two DNA fragments were purified,subcloned and sequenced.

In both cases, sequence analysis revealed an in frame fusion of novelsequences to HMGI-C. These sequences differed between the two lipomas,and immediately followed exon 3 of HMGI-C (FIG. 4).

The presence and specificity of chimeric transcripts in the two lipomaswere confirmed further by an independent RT-PCR. cDNA was prepared fromlipoma RNA samples but primers from the novel sequences, instead ofoligo-dT, were used for the reverse transcription reaction so that onlyRNA transcripts spanning the translocation would result in a PCRamplification product (FIG. 5). Products of the predicted size wereobserved only when primers derived from the novel sequences were used toreverse transcribe RNA isolated from the corresponding cell lines. Noproducts were seen in lipoma RNA from ST90-375 or ST93-724 when primers724 or 375 were used, respectively.

Finally, the chromosomal origin of the novel sequences was determinedusing DNA prepared from a monochromosomal rodent-human somatic cellhybrid panel. Specific primers were designed for the two novel sequencesobtained from the lipoma cDNAs. PCR performed on genomic DNA from thesomatic cell hybrids demonstrated that the novel sequence fused toHMGI-C in ST93-724, with a t(3;12), was located on chromosome 3 (FIG. 6)and the novel sequence from ST90-375, with a t(12;15), mapped tochromosome 15 (FIG. 6).

Novel Sequences Encode for Transcriptional Regulatory Domains

A detailed computer analysis of the novel sequences from the twoamplified fusion transcripts demonstrated that they encode putativetranscriptional regulatory domains. Inspection of the predicted proteinsequence from ST93-724 revealed the presence of two tandemly arrayed LIMdomains (Sanchez-Garcia and Rabbitts, 1993) separated by thecharacteristic 8-10 amino acids (FIG. 6A). These domains are 50-60 aminoacid residue motifs which are rich in cysteine and histidine and werefirst identified in three proteins, lin-11, Isl-1 and mec-3 (Way andChalfie, 1988; Freyd et al., 1990; Karlsson et al., 1990). The domain isorganized into two adjacent zinc fingers separated by a two residuelinker (Feuerstein et al., 1994) and members of the LIM family ofproteins may contain one or more LIM domains (S{acute over(a)}nchez-Garc{acute over (i)}a et al., 1993). Many of theLIM-containing proteins are transcription factors (S{acute over(a)}nchez-Garc{acute over (i)}a et al., 1993) and their activity isthought to be regulated by protein-protein interactions through theability of LIM domains to dimerize (Feuerstein et al., 1994).

Computer analysis of the novel sequence from ST90-375 did not reveal anysignificant homology with known sequences. Notably, the carboxy-terminalend of the predicted protein is highly acidic (pI 4.6) and rich inserine and threonine residues. Such domains have been implicated intranscriptional activation and have been shown to stimulatetranscription from remote as well as proximal positions (Mitchell andTijan, 1989; Seipel et al., 1992).

Therefore, the predicted domain organization of the wildtype HMGI-C andthe fusion proteins can be schematically depicted as shown in FIG. 7. Inboth fusion proteins, the C-terminal domain of the wildtype HMGI-C,which does not activate transcription (Thanos and Maniatis, 1992; X.Z.and K.C., unpublished data) is replaced by distinct, potentialtranscription regulation domains. These newly acquired functionaldomains in combination with the A-T hooks of HMGI-C would give rise tounique proteins that may contribute to the pathobiology of lipomas.

HMGI Proteins in Mammalian Growth and Development

Previous studies (King, J., 1955) had established that the pygmyphenotype could be observed at birth. Therefore, RNA from wildtype mouseembryos was isolated (Chirgwin, J. et al., 1979) and Northern blotanalysis revealed a transcript of 4.1 kb (FIG. 9). As expected from thegenomic analysis, no detectable HMGI-C expression was observed in thespontaneous and transgenic insertional mouse mutants. Additionally, athird allele exists at the pygmy locus (Green, M. C., 1989), In(10)17Rk,which carries an inversion of chromosome 10 and the distal breakpoint iswithin intron 3 of the HMGI-C gene (data not shown).

No HMGI-C expression was detected in homozygous embryonic In(10)17Rk RNA(FIG. 9). Quantitation by phosphorimager analysis revealed thatheterozygous mice expressed HMGI-C at approximately 50% wildtype levels.Therefore, the wildtype allele in the heterozygous mice does notincrease its expression levels to compensate for the loss of the deletedallele. This is consistent with the pygmy mutation being semi-dominantbecause there is a mild phenotypic effect on heterozygous mice (80% theweight of wildtype mice) (Benson, K. & Chada, K., 1994). Furthermore,HMGI(Y), the only other known member of the HMGI gene family(Grosschedl, R. et al., 1994), retained the same levels of expression inthe mutant and wildtype mice (FIG. 9). Therefore, there is nocompensation by HMGI(Y) for the lack of HMGI-C expression in pygmy mice.

The mutant alleles described above arise from major disruptions ofgenomic DNA which result in large deletions or a chromosomal inversion.To exclude the possibility that a gene other than HMGI-C may beresponsible for the pygmy phenotype, a mouse null mutant of HMGI-C wasproduced by targeted disruption. Mouse embryonic stem (ES) cells weregenerated that had 3.0 kb of the HMGI-C gene, encompassing exons 1 and2, replaced with a neomycin-resistance gene (FIG. 10(A)). Matingsbetween mice heterozygous for the mutated allele produced micehomozygous for the disrupted allele (FIG. 10(B)) at the expectedMendelian frequency of approximately 25% (13/51). Immunoblot analysisdemonstrated an absence of HMGI-C in protein extracts from homozygousembryos (FIG. 10(C)). Homozygous HMGI-C^(−/−) mice revealed theclassical features of the pygmy phenotype which include reducedbirthweight, craniofacial defects (shortened head) and an adult bodyweight of approximately 40% (39.8 +/−2.9) of wildtype littermates(Benson, K. & Chada, K., 1994). Therefore, it can be concluded thatabsence of HMGI-C expression in mice causes the pygmy phenotype.

Previously, a restricted number of adult tissues were analysed(Manfioletti, G. et al., 1991) and established that the endogenousexpression of HMGI-C could not be detected. Hence, a more comprehensivepanel of tissues were examined to investigate the temporal and tissuespecific expression pattern of HMGI-C. Within the sensitivity ofNorthern blot analysis, HMGI-C expression was not detected in 18 adulttissues (data not shown). However, expression of HMGI-C was observedduring mouse embryogenesis (FIG. 1(A)) as early as 10.5 days post coitum(dpc), but essentially disappeared by 15.5 dpc. Remarkably, the otherfamily member, HMGI(Y), showed a similar endogenous expression pattern(FIG. 11(A)) with expression readily observed in 10.5-16.5 dpc mouseembryos. The predominant expression of HMGI-C and HMGI(Y) duringembryogenesis suggests this architectural factor family functions mainlyin mammalian development.

The analysis of HMGI-C expression was further extended by itslocalization in the normal developing mouse embryo. Expression wasobserved in the majority of tissues and organs during embryogenesis asexemplified by the 11.5 dpc mouse embryo (FIG. 11(B)). Noticeably,HMGI-C expression was not seen in the embryonic brain except in a small,localized region of the forebrain (FIG. 11(B)). This expression patterncoincides with previous studies which demonstrated that most tissues inpygmy mice were 40-50% smaller as compared to wildtype tissues and theonly tissue of normal size was found to be the brain (Benson, K. &Chada, K., 1994).

To initiate studies on the elucidation of the role of HMGI-C in cellgrowth, embryonic fibroblasts were cultured from homozygous and wildtypeembryos. Strikingly, the number of pg/pg embryonic fibroblasts wasfour-fold less as compared to wildtype fibroblasts after four days invitro (FIG. 11(C)) and was not due to cell death. This data, as well assimilar studies in other systems (Ram, T. et al., 1993; Berlingieri, M.T. et al., 1995), is consistent with a role for HMGI-C in cellproliferation and suggests that HMGI-C functions in a cell autonomousmanner. Furthermore, absence of HMGI-C expression in the pygmy mutantwould then lead to a decrease in cell proliferation and causes thereduced size of all the tissues except for the brain.

Misexpression of Disrupted HMGI Proteins in Human Tumors

Isolation and Analysis of the Aberrant HMGI Transcripts

Rearrangements of HMGI-C in human tumors always preserve the DNA-bindingdomains of the protein and the DNA-binding activity of the HMGIarchitectural factors is essential for the enhancer activation.Moreover, sequence analysis demonstrated that the DNA-binding domainsare completely conserved between human HMGI-C and HMGI(Y) (Figure notshown). Therefore, HMGI expression was investigated in human tumors withkaryotypic abnormalities involving chromosomal band 6p21-23.

Establishment of cell lines is frequently associated with accumulationof mutations in vitro. To exclude such artifacts, RNA was isolateddirectly from frozen tumor samples. Lipomas ST92-24269 t(4;6) andST88-08203 t(6; 11) were karyotyped and total RNA was purified fromfrozen tissues by cesium chloride centrifugation. Next, amplification ofthe HMGI transcripts was performed using 3′ RACE protocol. Upon analysisof the resulting reactions by gel electrophoresis, aberrant HMGI(Y)products were readily detectable (Figure not shown). At the same time,HMGI-C expression was not detected in these tumors (Figure not shown).

The anomalous HMGI(Y) cDNAs were further characterized by sequenceanalysis. In lipoma ST92-24269, the transcript encoded for the 5′ end ofHMGI(Y) followed by a novel sequence (Figure not shown). Comparison ofthis latter sequence to the Genbank database revealed that it wasderived from the 3′ UTR of wild-type HMGI(Y). PCR analysis of thegenomic DNA from tumor ST92-24269 determined the transcript was producedby an internal deletion of both exonic and intronic sequences(unpublished data) which removed 922 bp from the wild-type HMGI(Y) cDNA(Figure not shown).

Sequencing of the aberrant transcript in lipoma ST88-08203 revealed afully intact HMGI(Y) open reading frame. A detailed molecular analysisdemonstrated that this transcript was produced by the removal of 923 bpof the wild-type sequence from exon 8 (Figure not shown). Interestingly,the rearrangement was limited to the 3′ UTR of the gene, leaving thecoding sequence intact. Therefore, the aberrant transcripts isolatedfrom the lipomas with rearrangements of 6p21 are produced by internaldeletions within the HMGI(Y) gene. The findings in both tumors wereconfirmed by an independent RT-PCR in which an HMGI(Y)-specific reverseprimer rather than oligo-(dT) was used for reverse transcription andsubsequent PCR (Figure not shown).

In lipoma ST92-24269, the predicted HMGI(Y) fusion protein consists ofthe first two DNA-binding domains of HMGI(Y) fused in frame to anuninterrupted open reading frame (ORF) encoding for 108 amino acidresidues. A detailed examination of the ORF revealed an unusually highcontent of proline (17%) which is indicative of a potentialtranscriptional regulatory domain (Figure not shown). Therefore, theoverall structure of this HMGI(Y) fusion protein is remarkably similarto proteins produced by disruptions at 12q13-15 which juxtaposedDNA-binding domains of HMGI-C to putative transcriptional regulatorydomains.

Translation of the HMGI(Y) aberrant transcript in the tumor ST88-08203predicted a normal protein. In contrast, in previously described lipomaschimeric HMGI-C transcripts encoded for novel fusion proteins whoseformation was proposed to be necessary for lipoma development. Toestablish whether differences in the overall domain organization of theHMGI(Y) and HMGI-C fusion proteins found in lipomas are due to thedistinct properties of these two genes, an additional tumor with HMGI-Crearrangement, lipoma ST91-198 t(12;15), was analyzed. RNA was isolatedfrom the primary cell culture and 3′ RACE used to amplify the HMGI-Cchimeric transcript (unpublished data). The molecular analysis of thiscDNA revealed that it preserved the first three exons of HMGI-C thatencode for the HMGI DNA-binding domains. However, the endogenous HMGIexons four and five were removed and replaced by a heterologous sequence(Figure not shown). Notably, an in-frame stop codon present in thissequence terminates translation of the chimeric transcript after addingonly ten amino acid residues to the HMGI-C DNA-binding domains. Thesequence of the novel peptide did not contain any distinguishingfeatures and revealed no significant homology with known proteins.Chromosomal rearrangement in tumor ST91-198 therefore results in atruncated protein that consists mainly of the HMGI-C AT-hooks.Accordingly, a simple truncation of either HMGI(Y) or HMGI-C issufficient to cause lipomas.

Lipomas Can Bypass Expression of the Wild-type HMGI Allele Expression ofthe wildtype HMGI proteins is highly associated with transformation andcan be detected in a wide variety of tumors. Moreover, inhibition ofHMGI-C synthesis was shown to render several distinct cell typesintransigent to retroviral transformation, suggesting that HMGIexpression is required for tumorigenesis. Appreciable levels ofwild-type HMGI(Y) expression that were found in tumor ST88-08203(Figures not shown) are in agreement with this hypothesis. Surprisingly,the non-rearranged allele was not expressed in lipoma ST92-24269(Figures not shown) where an HMGI(Y) fusion protein was identified.

In lipomas with rearrangements of 12q13-15, chimeric transcripts areproduced by the juxtaposition of HMGI-C with the heterologous sequencesand therefore cannot be readily amplified in the same PCR reaction withthe wildtype cDNA. To assess the expression of the wild-type HMGI-C inthese tumors, the highly polymorphic microsatellite sequence located inthe 5′ UTR of HMGI-C was employed. Oligonucleotide primers complimentaryto the sequences flanking this polypyrimidine tract were synthesized andused for RT-PCR (Figure not shown). Again, expression from thenon-rearranged allele was only observed in the tumor with a truncatedHMGI-C. No expression of the wildtype allele but not in the lipomaST93-724, in which HMGI-C DNA-binding domains were fused to LIM domains,motifs that function in transcriptional regulation (Figure not shown).

Differentiated Adipocytes Express HMGI-C in Lipomas but not in NormalFat

During development, the expression of the HMGI proteins is tightlyregulated. HMGI expression is found in the developing tissues and organsof the mouse embryo but essentially dissapears by the end ofintrauterine development and can no longer be found postnatally. Toconfirm that HMGI expression in lipomas is not a consequence of theendogenous HMGI expression by the adult adipose tissue,immunocytochemistry was performed with an antibody raised againstHMGI-C. In full agreement with numerous previous findings whichdemonstrated that HMGI proteins are not expressed by differentiatedcells or adult tissues, HMGI-C expression could not be detected in theadult adipose tissue (Figure not shown). Futhermore, RT-PCR with primersspecific for HMGI-C and HMGI(Y) confirmed that HMGI genes are notexpressed in normal fat (unpublished data). However, the majority ofdifferentiated adipocytes in these neoplasms stained positively forHMGI-C (Figure not shown). Overall, HMGI-C expression was detected in75% (22 out of 29) of tumors (unpublished data).

Translocation Breakpoints Upstream of the HMGI-C Gene in UterineLeiomyomata

FISH analysis was performed on metaphase cells from uterine leiomyomatawith chromosome 12 rearrangements (Table not shown) by use of clonesfrom the 5′ and 3′ ends of HMGI-C (Figure not shown). In contrast tolipomas, where translocation occurs in frame following exon 3, both 5′and 3′ clones hybridized only, on the rearranged chromosome not derivedfrom chromosome 12, in addition to the normal 12 homolog, indicatingthat the entire sequence encoding HMGI-C maps distal to thetranslocation breakpoint. In two uterine leiomyomata with typicalt(12,14) translocations (ST90-194 and ST93-738), breakpoints mappedwithin the same lambda clones approximately 10 kb upstream of exon 1 ofHMGI-C (Figures not shown). These breakpoints were verified by Southernblot analysis, a 3.3 kb probe from lambda clone H528 detected rearrangedbands in both tumors (unpublished data). In ST92-224, a uterineleiomyoma with a variant translocation involving chromosome 1, thebreakpoint mapped within this same region, indicating that this site onchromosome 12 contains a critical region for rearrangement regardless ofthe chromosomal origin of the translocated material. FISH analysis ofST94-114, another uterine leiomyoma with a characteristic t(12;14)revealed a breakpoint approximately 100 kb 5′ of HMGIC. In two otheruterine leiomyomata (ST93-165 and ST89-171), breakpoints occurred morethan 100 kb upstream of HMGI-C as the most 5′ lambda clone in the contig(H121) is translocated to the der(14) chromosome in these tumors.ST89-171 contains two normal chromosome 12 homologs in addition to ader(14)t(12;14); therefore, hybridization signals corresponding to threecopies of HMGI-C were detected (Figure not shown). Finally, anotheruterine leiomyoma (ST93-220) with an atypical cytogenetic rearrangementin which the involved segment of chromosome 12 appeared to be proximalin band q13 was determined by FISH to have a deletion startingapproximately 10 kb upstream of HMGI-C and extending up to about 100 kb5′ of exon 1 of HMGI-C (Figure not shown).

DISCUSSION HMGI Proteins in Adipogenesis and Mesenchyme Differentiation

In this study, chimeric transcripts were identified from two lipomaswhich resulted from fusion of the 5′ end of the HMGI-C gene to novelsequences derived from different chromosomes. Three DNA binding domainscontaining the A-T hook motifs of HMGI-C are linked in these transcriptsto sequences encoding potential transcriptional regulatory domains. Inthe case of lipoma ST90-375, the novel domain is highly acidic and richin serine and threonine residues resembling the typical activationdomains found in transcription factors. In lipoma ST93-724, the novelprotein contains two LIM domains, motifs that promote protein-proteininteractions.

HMGI-C, Chimeric Transcripts and Lipomas

The chromosomal region 12q14-15 is hypothesized to contain an importantgene involved in lipomas because it is the most commonly rearranged site(Mandahl et al., 1988). Our study establishes that HMGI-C is the genedisrupted in lipomas with chromosome 12 rearrangments. The large intron(greater than 25 kb) between exons 3 and 4 distinctly separates theDNA-binding from the acidic domains of HMGI-C. This provides asubstantial target for translocations so that the three A-T hook motifsremain intact and confer the DNA-binding specificity of HMGI-C to thefusion proteins.

HMGI-C is a 109 amino acid residue protein (Patel et al., 1994) thatconsists of three DNA-binding domains (A-T hooks) linked to thecarboxy-terminal acidic domain which does not activate transcription(Thanos and Maniatis, 1992; X. Z. and K. C., unpublished data). The twolipoma translocations result in a novel protein containing A-T hooks ofHMGI-C at the amino-terminus fused to transcriptional regulatory domainsat the carboxy end. The other reported example for an A-T hookcontaining gene implicated in tumorigenesis is MLL (Tkachuk et al.,1992; Gu et al., 1992). However, the presence of a putative second DNA20binding domain (Ma et al., 1993) derived from the MLL gene and retainedin the fusion protein obscures the exact contribution of the A-T hooksto tumor pathogenesis (Rabbitts, 1994). In these lipomas, the only knownHMGI-C functional domains retained in the fusion proteins are the A-Thooks. These motifs would therefore be responsible for DNA bindingspecificity of the fusion proteins. Although it is possible that simpletruncation of HMGI-C is sufficient to cause lipomas, a number of studieshave determined that both domains of fusion proteins are necessary fortransforming activity (de The et al., 1991; Kamps et al., 1991;

Pendergast et al., 1991; May et al., 1993). Therefore, as proposed forother fusion proteins, the heterologous sequence in the lipoma fusionproteins would alter the biological activity of wildtype HMGI-C and leadto deregulation of downstream target genes.

The above model readily explains how the fusion protein produced inlipoma ST90-375 may function. The novel sequence from chromosome 15encodes for an acidic peptide rich in serine and threonine residues.These features have been observed in a number of transcriptionalactivation domains (Mitchell and Tijan, 1989) including thecarboxy-terminal domains of homeobox proteins (Hatano et al., 1991) andNF-KB (Schmitz and Baeuerle, 1991). So, the acquisition of atransactivation domain by the DNA-binding domains of HMGI-C, whichnormally possesses a transcriptionally inactive acidic domain, caneasily be reconciled with aberrant regulation of the HMGI-C targetgenes. In the case of the t(3;12) in ST93-724, the fusion protein mustoperate by a different mechanism to deregulate the HMGI-C target genes.The novel sequence from chromosome 3 encodes for two tandemly arrangedLIM motifs. The LIM domain is conserved amongst highly diverged speciesand LIM proteins have been shown to have important developmentalfunctions which include patterning (Cohen et al., 1992), cell fatedecision (Freyd et al., 1990) and differentiation (Way and Chalfie,1988). LIM domain proteins are capable of protein-protein interactions(Sadler et al., 1992) through dimerization mediated by the LIM domains(Feuerstein et al., 1994). Therefore, LIM-LIM interactions between theST93-724 fusion product and other nuclear proteins could recruitpotential transcriptional regulators to DNA sequences with a specificitydictated by the HMGI-C A-T hook motifs. Deregulation of HMGI-C targetgenes would then contribute to lipoma development. It is interesting tonote that the majority of nuclear proteins capable of interacting withLIM domains are known to function as transcription factors. Theseinclude several LIM-homeodomain proteins (Sanchez-Garcia et al., 1993and references within) as well as basic helix-loop-helix proteinsshPan-1 (German et al., 1992) and TAL1 (Valge-Archer et al., 1994).While overexpression of LIM proteins has been implicated in T-celllymphomas (reviewed by Sanchez-Garcia and Rabbits, 1993), this is thefirst example of a LIM domain occurring in a fusion product.

A great heterogeneity in chromosomal partners translocated with 12q14-15is found in karyotypically abnormal lipomas indicating that a largenumber of sequences in the genome can be fused to HMGI-C. The presentdata demonstrate that novel sequences linked to HMGI-C in two lipomasencode for distinct domains. This suggests that a number of alternativedomains can be placed downstream of the HMGI-C A-T hooks and contributeto the pathobiology of lipomas. Interestingly, both novel sequencesdescribed in this study encode for transcriptional regulatory domains.Therefore, the choice of novel sequences in chimeric transcripts inlipomas is presumably not arbitrary but does require the presence ofregulatory transcriptional domains attached to the HMGI-C A-T hooks.

A similar situation has been observed in the 11 q23 acute leukemiaswhere the MLL gene is translocated with multiple chromosomal partnerswhich mostly encode different types of transcriptional regulatorydomains (Prasad et al., 1994). This could be a general mechanism fortumors where nonrandom rearrangements of a specific chromosomal regioninvolve a variety of partners. Chimeric transcription factors thatpromote tumorigenesis would be produced by juxtaposing DNA bindingdomain(s) contributed by the consistently rearranged locus to distincttypes of transcriptional regulatory domains.

HMGI-C, Pygmy and Adipogenesis

The above studies demonstrate that an altered HMGI-C protein is involvedin the abnormal growth and development of fat cells resulting inlipomas. This leads to the possibility that HMGI-C may normally play arole in adipogenesis and analysis of the pygmy mouse stronglysubstantiates this hypothesis. The mouse mutant, pygmy, was found to bea null mutation of HMGI-C due to deletions within the gene (unpublishedresults). The obvious phenotypic characteristics of the pygmy mouse areits small stature with most tissues reduced commensurate with theoverall decrease in weight of the mouse (40% of wildtype).Interestingly, one tissue disproportionately reduced in weight is bodyfat. The fat index, a reliable indicator of total fat content relativeto body weight (Rogers and Webb, 1980), is approximately eight timeslower in pygmy than in their wild-type littermates (Benson and Chada,1994). The function of HMGI-C in adipogenesis could be related to itsrole in cells undergoing differentiation. It is expressed in lessdifferentiated cells but no detectable levels are observed in theirterminally differentiated counterparts (Vartainen et al., 1988;Giancotti et al., 1987).

Therefore, lack of HMGI-C expression, as found in the pygmy mouse, couldaffect the differentiation of preadipocytes into mature adipocytes,cells capable of lipid storage. This developmental abnormality wouldlead to a decrease in fat deposition and the phenotype observed in thepygmy mouse. The role of HMGI-C in adipogenesis and metabolic disorderssuch as obesity is thus of considerable interest.

Relevance of HMGI Family in Tumors with Rearrangements of 12q13-15 or6p21

Of major importance is the frequent observation of chromosomalrearrangements in bands 12q13-15 in a large group of benign solidtumors. Most prominently, these include uterine leiomyomata (Nilbert andHeim, 1990; Rein et al., 1991), and pleomorphic adenomas of the salivarygland (Sandros et al., 1990; Bullerdiek et al., 1993). Rearrangements of12q13-15 have also been reported in pulmonary chondroid hamartomas (DalCin et al., 1993; Fletcher et al., 1995), endometrial polyps (Vanni etal., 1993), epithelial breast tumors (Rohen et al., 1993),hemangiopericytoma (Mandahl et al., 1993a), chondromatous tumors(Mandahl et al., 1989, 1993b; Bridge et al., 1992; Hirabayashi et al.,1992), diffuse astrocytomas (Jenkins et al., 1989), parosteal lipoma(Bridge et al., 1995), and a giant-cell tumor of the bone (Noguera etal., 1989). Many of these tumor types are of mesenchymal origin and ithas therefore been hypothesized that a single gene associated withgrowth and mesenchyme may be responsible for these multiple neoplasms(Schoenberg Fejzo et al., 1995). Several lines of evidence implicateHMGI-C as a strong candidate for such a gene at 12q14-15. First,physical mapping studies have shown chromosomal breakpoints for three ofthese benign tumors (lipoma, pulmonary chondroid hamartoma and uterineleiomyoma) to map within a single YAC (Schoenberg Fejzo et al., 1995).This study assigns HMGI-C to the translocation breakpoint in lipomas,and chromosomal breakpoints in five analyzed uterine leiomyomata as wellas a pulmonary chondroid hamartoma have been found to reside within10-100 kb of exon 1 of HMGI-C (unpublished results). Second, the role ofHMGI-C in growth control is apparent because its disruption in the pygmymouse leads to aberrant growth and development. Also, it has been shownin vitro that HMGI-C is required for transformation (Berlingieri et al.,1995). Finally, preliminary studies reveal that expression of HMGI-Cduring mouse embryogenesis is restricted mainly to the mesenchymalcomponent of tissues and organs (unpublished results). Taken together,these data indicate that HMGI-C is highly likely to be the genedisturbed by 12q14-15 rearrangements in a number of tumors ofmesenchymal origin.

Nonrandom involvement of 6p21-23 has also been observed in lipomas(Sreekantaiah et al., 1991), pulmonary chondroid hamartomas (Fletcher etal., 1992, 1995) and uterine leiomyomata (Nilbert et al., 1990).Interestingly, HMGI(Y), the other member of the HMGI protein family witha similar structure as HMGI-C that includes the three DNA-bindingdomains, has been localized to 6p21 (Friedmann et al., 1993). Thisraises an intriguing possibility that HMGI(Y), a molecule closelyrelated to, but distinct from HMGI-C, could also be associated withbenign tumors of mesenchymal origin.

In summary, a disruption of the HMGI-C gene resulting in chimerictranscripts is a characteristic feature of lipomas. As adipocytes play akey role in lipid homeostasis and maintenance of energy balance invertebrates, an understanding of HMGI-C function in adipogenesis maylead to insights into obesity and other metabolic disorders. Inaddition, the obvious role of HMGI-C in normal growth demonstrated bythe phenotype of the pygmy mouse and its localization at or adjacent tothe translocation breakpoints in lipoma, uterine leiomyoma and pulmonarychondroid hamartoma suggests its fundamental involvement in a variety ofbenign tumors.

HMGI Proteins in Mammalian Growth and Development

The current study demonstrates that the absence of HMGI-C causes growthretardation in pygmy mice. Although the precise molecular mechanismremains to be elucidated, the function of HMGI proteins in cellproliferation could be regulated during the cell cycle throughalteration of their DNA binding ability via phosphorylation by the cellcycle-dependent p34cdc2 kinase (Reeves, R. et al., 1991). Inactivationof the HMGI-C gene would perturb the cell cycle in the developing embryoand the resulting disruption of growth would produce the pygmyphenotype. The identification of the pygmy gene as HMGI-C provides novelinsights into the control of mammalian growth and development and amolecular clue to investigate the biochemical nature of the Africanpygmy phenotype (Sinha, Y. et al., 1979) and a multitude of growthhormone-resistant human dwarf syndromes (Benson, K. & Chada, K., 1994).

Misexpression of Disrupted HMGI Proteins in Human Tumors

HMGI(Y) and HMGI-C, two homologous but distinct members of the HMGIfamily of architectural factors, have now been shown to be disrupted inidentical tumors. Rearrangements of HMGI-C, first reported in lipomas,were later described in other mesenchymally derived neoplasms withtranslocations of 12q13-15. Similar to HMGI-C, disruptions of HMGI(Y)will presumably be also responsible for uterine leiomyoma, pulmonaryhamartoma, pleomorphic adenomas of salivary gland and other mesenchymaltumors with recurrent aberrations at 6p21-23.

Rearrangements within HMGI Genes are Required for Lipoma Development

In combination with previous studies on HMGI(C) and HMGI-Y, it is nowpossible to glean novel insight into the molecular mechanism of tumorformation in lipomas and, by extrapolation, in related solid mesenchymalneoplasms. HMGI-C does not behave as a classical transforming oncogenesince overexpression of full-length HMGI-C cDNA does not result intumorigenesis. On the other hand, in all twelve analyzed lipomas,chromosomal rearrangements have produced disruptions in translocatedHMGI alleles. While expression of an HMGI gene is necessary fortumorigenesis, activation of an intact HMGI allele in a mesenchymal cellwill not be sufficient to produce a tumor. Therefore, disruptions withinHMGI genes and the aberrant structure of the resulting cDNA are requiredfor lipoma development.

A variety of the HMGI chimeric transcripts can be found in lipomas. Thecomparison of these aberrant cDNAs demonstrates that rearrangements canrange from a simple internal deletion to protein truncation tojuxtaposition of transcriptional regulatory domains to HMGI DNA-bindingdomains. An aberration common to these twelve lipomas is a deletion ofor within highly conserved and unusually large and 3′ UTR of an HMGIgene. The best example is lipoma ST8808203, where the aberranttranscript codes for the wild-type HMGI(Y) and the deletion is limitedto its 3′ UTR. Since translocations of 12q13-15 which disrupt 3′ UTR ofHMGI-C while preserving its ORF are also observed in leiomyoma andpleiomorphic adenoma of salivary gland, 3′ UTRs of HMGI genes maycontain important regulatory sequences that function in growthregulation and/or tumor suppression.

Notably, the aberrant transcripts isolated from lipomas withrearrangements of 6p21-23 were generated by internal deletions withinthe translocated HMGI(Y) allele. This observation suggests that inlipomas and related benign mesenchymal tumors, HMGI genes may containinternal deletions and other submicroscopic rearrangements undetectableby cytogenetic techniques. It is likely therefore that the contributionof the HMGI genes to tumorigenesis is more significant than predicted bykaryotypic analysis.

Misexpression of HMGI genes in a Differentiated Cell Results inTumorigenesis

To understand the biological function of the HMGI proteins, it isimportant to analyze their expression profiles during both normal andpathological growth. Prominently, high levels of the HMGI expression areobserved during mouse embryonic development in midgestation but itessentially dissappears closer to the end of pregnancy. Subsequently, noHMGI expression can be detected in any of the adult tissues. Lipomas arecomposed of mature adipocytes which, like other terminallydifferentiated cells, normally do not express HMGI proteins. However,transcriptionally active HMGI alleles are consistently found in solidmesenchymal tumors with rearrangements of 12q13-15 and 6p21-23.Rearrangements of 12q13-15 or 6p21-23 activate an HMGI allele normallysilent in adult cells and the resulting misexpression of the HMGIprotein in the context of a differentiated mesenchymal cell is a crucialstep in tumor development. A notable feature of this mechanism stemsfrom the observation that during mouse embryonigenesis, HMGI-C isexpressed in the mesenchymal component of the developing organs andtissues (unpublished data). Tumorigenesis in this case results from thetemporally inappropriate expression in an adult cell of a gene that isnormally expressed during prenatal development in an embryonic cell ofthe same lineage. This is reminiscent of observations in B-cellleukemias where rearrangements of 8q24 chromosomal area activate c-mycexpression in a precursor cell of B-lineage and result in neoplasia.Unlike the HMGI family members, however, the endogenous expression ofc-myc is not restricted to embryogenesis and its inappropriateexpression takes place at the same time in the life of the organism whenit is normally expressed. Even more different is a situation in some ofthe T-cell acute lymphoid leukemias where the cause of neoplasia isectopic expression in T-cell precursors of HOX11, normally expressed inthe embryonic liver.

Distinct Molecular Pathways of Tumorigenesis Exist in Lipomas

The molecular analysis of the lipomas described above yields valuableinformation about the expression state of the non-rearranged HMGIalleles. Wildtype HMGI expression, normally associated withtumorigenesis, was readily detectable in lipomas ST88-08203 andST91-198, where chromosomal rearrangements produced an apparently normalHMGI(Y) and a truncated HMGI-C proteins, respectively. In contrast, thenon-rearranged HMGI allele was not expressed in tumors ST92-24269 andST93-724, where the aberrant HMGI transcripts were predicted to encodefusion proteins consisting of the HMGI DNA-binding domains fused toputative transcriptional regulatory domains.

The above findings indicate that there are at least two distinctmolecular pathways by which tumorigenesis in lipomas can proceed. When achromosomal rearrangement produces a disrupted HMGI protein with nointrinsic transcriptional activity, tumor development is dependent uponsubsequent activation of the non-rearranged allele. However, therequirement for wildtype HMGI expression can be circumvented when, as aresult of a translocation, a transcriptional regulatory domain isjuxtaposed to the HMGI AT-hooks. The unlikely alternative mechanism, inwhich the non-rearranged allele is activated by the fusion proteinthrough a positive HMGI regulatory mechanism, would postulate that suchautoregulatory function is inhibited in the presence of transcriptionalregulatory domains. Therefore, we conclude that distinct rearrangementsof a single gene can activate alternative molecular pathways of tumorpathogenesis.

Molecular analysis of HMGI rearrangements in multiple tumor samples cannow be combined with the expression studies of both disrupted andnon-rearranged alleles to produce a mechanistically coherent model oflipoma development (Figure not shown). Tumor development is initiatedwhen the chromosomal rearrangement disrupt an HMGI allele and results inthe HMGI misexpression in a differentiated mesenchymal cell. Deletionwithin 3′ UTR is probably the minimal rearrangement necessary for tumorformation. Subsequently, one of the alternative tumorigenic pathways isselected based on the precise nature of the HMGI disruption. In thesimplest model, the requirement for HMGI expression in tumorigenesiscould be circumvented if HMGI DNA-binding domains are juxtaposed with atranscriptional regulatory domain (Figure not shown). The reduced numberof events involved in tumor formation would readily explain the mostfrequently observed translocation in lipomas, t(3;12)(q29;q15), since itfuses DNA-binding domains of HMGI-C with LIM domains, motifs that arethought to function in transcriptional regulation.

The HMGI Proteins Play Different Roles in Tumors of Epithelial andMesenchymal Origin

Benign tumors, unlike their malignant counterparts, are characterized bya limited number of highly specific genetic alterations involving only afew chromosomal regions. It was proposed therefore that the molecularanalysis of these neoplasms would identify genes of major importance forgrowth and proliferation. The above studies with HMGI(Y) and HMGI-C inlipomas demonstrate that misexpression of HMGI proteins plays asignificant role in the development of a diverse array of human solidtumors. Clinically, a prominent feature of these benign mesenchymaltumors is the extremely low rate at which they convert to malignancy.Indeed, uterine leiomyomas progress to become leiomyosarcomas in lessthan 0.01% of the cases while conversion of lipoma to liposarcoma iseven less frequent. Therefore, misexpression of HMGI proteins, whileacting to increase the growth rate of the mesenchymal cells, does notseem to predispose the overproliferating cell to malignanttransformation and may even play a protective role.

The apparent inability of the HMGI-expressing benign mesenchymal tumorsto undergo malignant conversion is in a stark contrast with thesituation seen in the tumors of epithelial origin. In these latterneoplasms, cellular hyperproliferation provides starting population forclonal expansion which, in turn, is followed by a stepwise progressionto malignancy. Even more intriguingly, epithelial cells cannot betransformed by overexpression of HMGI-C while chromosomal rearrangementswhich could disrupt HMGI-C and HMGI(Y) are not found in tumors of theepithelial origin. Finally, in epithelial tumors activation of HMGIexpression is associated with the advanced stages of carcinogenesisrather than with early hyperplasia. The asynchrony between theexpression patterns of HMGI proteins in epithelial and mesenchymal cellsas well as distinct phenotypes of the relevant tumors indicate that intissues of different embryonic lineage HMGI proteins perform dissimilarfunctions.

One possible explanation for this phenomenon is provided by the factthat HMGI proteins normally function in the developing mesenchyma. Therole of HMGI proteins in mesenchymal tumorigenesis may therefore beclosely related to that during normal development, such as growth rateregulation. In the epithelial tumors, the HMGI architectural factors,expressed outside of their normal cellular milieu, may be recruited totake part in the transcriptional regulation of genes that are involvedspecifically in the final stages of tumor progression, such as invasionand metastasis. Regardless of the molecular details, the ability ofHMGIC and HMGI(Y) to execute distinct functions during tumorigenesis indiverse cell types provides a powerful testimony to the biologicalpotency of the HMGI proteins and accounts for the dramatic consequencesof their disruption.

Translocation Breakpoints Upstream of the HMGI-C Gene in UterineLeiomyomata

Translocation breakpoints in uterine leiomyomata reported here are instark contrast to those observed in lipomas and other benign mesenchymaltumors in which translocations are found within the coding region ofHMGI-C. Unlike the findings in uterine leiomyomata rearrangements inlipomas consistently result in disruption of HMGI-C, whereby DNA-bindingA-T hook domains are separated from the 3′ region of the gene. BecauseHMGI-C has no transcriptional activation domain (unpublished data), thepathobiology of lipomas appears to result from juxtaposition of director indirect activation domains with the DNA-binding A-T hook domains,although an alternative explanation of truncation of the protein cannotbe ruled out at present.

These studies of uterine leiomyomata suggest a completely differentmolecular mechanism because the entire gene appears to be retained,suggesting that both the 5′ DNA-binding domain and the 3′ domain ofunknown function are necessary. The finding that chromosomalrearrangements were located 10 to >100 kb upstream of HMGI-C in sevenuterine leiomyomata suggests that breakpoints might disrupt regulatoryelements and alter the normal expression of HMGI-C, analogous to Burkittlymphoma, where translocations up to 100 kb upstream of MYC result inaberrant expression and neoplasia.

This “regulatory hypothesis” is supported by cytogenetic and FISHresults for the karyotypically variant uterine leiomyoma ST89-171. Inthis tumor, three copies of HMGIC were present, suggesting a dosagemechanism for altered expression levels. Additionally, loss of theder(12) chromosome in ST89-171 provides further evidence that theder(14) chromosome, to which HMGI-C maps, contains the criticalsequence.

This observation of an interstitial deletion upstream of HMGI-C in oneuterine leiomyoma with a variant rearrangement of chromosome 12 isimportant for the cytogenetic and molecular interpretation ofrearrangements in uterine leiomyomata and other tumors. This findingimplies that uterine leiomyomata with unusual cytogenetic rearrangementsof chromosome 12, and possibly other mesenchymal neoplasms withoutmicroscopically detectable chromosome 12 rearrangements, may havesubmicroscopic rearrangements of a critical region upstream of HMGI-C.Characterization of HMGI-C expression in uterine leiomyomata of allcytogenetic subgroups is now warranted for a more complete understandingof the pathobiologic mechanism.

Furthermore, this interpretation of a mechanism for dysregulation ofHMGI-C in uterine leiomyomata is substantiated by observation of arearrangement in a fibroid involving chromosomes 8 and 12 in which the3′ UTR of HMGI-C is disrupted. Such a rearrangement results similarly inretention of the entire coding region of HMGI-C, a finding previouslynoted in variant translocations in Burkitt lymphoma. However, thistranslocation breakpoint mapping in uterine leiomyomata and thederegulation model differ largely from that reported by others in whichintragenic breakpoints were found for some fibroids perhaps reflectingthe relatively limited number of tumors analyzed. Alternatively,although there are no data to support the existence of alternative 5′exons of HMGI-C or other uncharacteristic genes in the region, suchpossibilities, which might be affected by chromosomal rearrangement andcontribute to tumor biology, cannot be excluded. Regardless, a mechanismof dysregulation not involving a fusion transcript must be consideredfor tumors without intragenic rearrangements of HMGI-C becauseirrefutable data implicate HMGI-C as the critical gene in benignmesenchymal tumors with rearrangements of 12q14-15.

These findings are consistent with accumulating evidence for a primaryrole of HMGI-C in normal growth and differentiation of a variety oftissues. Besides expression of fusion transcripts in lipomas and otherbenign mesenchymal tumors and in mesenchymal components of tissues inthe developing mouse embryo, expression of HMGI-C is found only in cellsafter they become transformed and has been found to be necessary, butnot sufficient, for transformation. These studies indicate that HMGI-Calso may be deregulated through translocation in uterine leiomyomatawithout involvement of a fusion transcript.

The present invention is further illustrated by the following exampleswhich are not intended to limit the effective scope of the claims. Allparts and percentages in the examples and throughout the specificationand claims are by weight of the final composition unless otherwisespecified.

EXAMPLES HMGI Proteins in Adipogenesis and Mesenchyme Differentiation

The GenBank accession numbers for the novel sequences in the chimerictranscripts from ST90-375 and ST93-724 are U28131 and U28132,respectively.

Isolation of YACs at the Human Pygmy Locus

Initially, conserved fragments were isolated from the cloned, mousepygmy locus (Xiang et al., 1990; K. Benson and K. C., unpublishedobservations) and were used as probes on a normal, human lambda genomiclibrary (Sambrook et al., 1989). The cross-hybridizing clones wereisolated and relevant homologous fragments were subcloned and sequenced.Specific oligonucleotide primers (sequence 5′-AGGGGACAACAAATGCCCACAGGSEQ ID NO: 1 and 5′-CGTCACCAGGGACAGTTTCACTTGG SEQ ID NO: 2) weresynthesized and used to screen a human total genomic YAC library by thePCR-based method (Green and Olson, 1990). Four positive clones ofSaccharomyces cerevisiae containing YACs yWPR383, yWPR384, yWPR385 andyWPR386 were isolated.

Construction and Screening of Phage Libraries

High molecular weight DNA was isolated from yeast strains harboring YACsyWPR383 and yWPR384 (Guthrie and Fink, 1991), and partially digestedwith Sau3A. After partial fill-in of the Sau3A site, DNA was subclonedat the partially filled XhoI site of the predigested lambda FIXII vector(Stratagene, La Jolla, Calif.) and packaged in vitro (GIGAPACK IIpackaging extract, Stratagene). To select clones derived from the humanYACs, 6000 plaques from each library were probed with total humangenomic DNA and hybridizing plaques were spotted on plates inoculatedwith SRB(P2) cells in a gridded array. After incubating the plates at39° C. for 12 hours, plaques were transferred onto DURALON (Stratagene)membranes. These grids were used for identifying lambda clones thatcontained human HMGI-C exons by probing with mouse HMGI-C cDNA(unpublished results), using the same hybridization conditions asdetailed below for Southern analysis. Overlaps between contiguous clonesand colinearity with the genome were confirmed by a combination of cloneto clone and clone to genomic hybridizations along with restrictionmapping.

Southern Blot Analysis

10-12 mg of human DNA was digested with the appropriate restrictionenzymes, products resolved on 0.8% agarose gels and transferred ontoDURALON (Stratagene) membranes. Blots were treated with prehybridizationsolution (50% formamide, 5× SSC, 10× Denhardt's solution, 0.05M sodiumphosphate pH 6.8, 0.001M EDTA, 0.01 mg/ml denatured salmon sperm DNA,and 0.2% SDS) for 2 hours at 42° C. Probes were added to thehybridization solution (50% formamide, 5× SSC, 1× Denhardt's solution,0.02M sodium phosphate pH 6.8, 0.001M EDTA, 0.01 mg/ml denatured salmonsperm DNA, 0.2% SDS and 10% dextran sulfate) and hybridization wasperformed for 16 hours at 42° C. Membranes were washed with 2× SSC,0.001M EDTA, 0.5% SDS, 0.05% NaPPi and 0.01M sodium phosphate pH 6.8, at65° C. for 3× 1 hour periods and exposed to X-ray film at −70° C. withintensifying screens.

Identification and Characterization of Chimeric Transcripts

First strand cDNA was synthesized in a 20 ml reaction using an anchoredoligo-dT primer 5′-GCAATACGACTCACTATAG(T)₁₃ SEQ ID NO: 3 and SuperscriptII RT reverse transcriptase (BRL, Gaithersburg, Md.) according to themanufacturer's protocol. Primers used in the first round of 3′ RACE(Ausubel et al., 1989) were an HMGI-C exon 1 sense primer5′-CTTCAGCCCAGGGACAACC SEQ ID NO: 4 and an antisense adapter primer5′-GCAATACGACTCACTATAG SEQ ID NO: 5. One ml of first-strand cDNA wascombined with 25 pmole of sense primer in a 50 ml reaction mixture (60mM Tris-SO₄ (pH 9.1 at 25° C.); 18 mM (NH₄)₂SO₄; 2 mM MgSO₄; each dNTPat 200 mM; 2.5 U of Taq DNA polymerase (BRL)), denatured for 2 minutesat 94° C. and subjected to 5 cycles of linear amplification (Rother,1992) using the following conditions: 94° C., 30 seconds; 58° C., 20seconds; 72° C., 1 minute 30 seconds. Ten pmole of antisense primer werethen added and 25 cycles of exponential amplification were performed(94° C., 30 seconds; 56° C., 30 seconds; 72° C., 1 minute 30 seconds).

One ml of the PCR reaction was reamplified for 20 cycles with a nestedHMGI-C sense primer spanning exon 1 and 2, 5′-GGAAGCAGCAGCAAGAACC SEQ IDNO: 6 as described above. Five ml of each reaction were analyzed on a1.5% agarose gel. Reverse transcription for the detection of chimerictranscripts using novel sequence-specific primers was performed as aboveexcept primers 375 (5′-CTTCTTTCTCTGCCGCATCG SEQ ID NO: 7) for ST90-375and 724 (5′-GTGAGGATGATAGGCCTTCC SEQ ID NO: 8) for ST93-724 were used.Subsequent PCR conditions were an initial denaturation at 94° C. for 2minutes; 30 cycles at 94° C., 30 seconds; 58° C., 30 seconds; 72° C., 1minute, followed by a final extension for 10 minutes at 72° C.

Chimeric transcripts amplified by 3′-RACE and RT-PCR were isolated fromthe gel, blunt-end cloned by standard methods (Sambrook et al., 1989)into the pCR-Script vector (Stratagene) and sequenced using theSequenase kit Version 2.0 (USB, Cleveland, Ohio).

Chromosomal Localization of Novel Sequences

The NIGMS monochromosomal somatic cell hybrid mapping panel #2 wasobtained from the Coriell Cell Repositories (Coriell Institute forMedical Research, Camden, N. J.). Primers used were derived from thenovel sequences of the chimeric transcripts and 500 ng of genomic DNAfrom each somatic cell line was used as a template for PCRamplification. For the novel sequence derived from the chimerictranscript obtained from lipoma ST90-375, the primers were5′-CAGAAGCAGACCAGCAAACC SEQ ID NO: 9 and 5′-CTTCTTTCTCTGCCGCATCG SEQ IDNO: 10 and from lipoma ST93-724, the primers were5′-CTCTGGAGCAGTGCAATGTG SEQ ID NO: 11 and 5′-GTGAGGATGATAGGCCTTCC SEQ IDNO: 12. PCR conditions for the ST93-724 novel sequence primers were 26cycles of 94° C., 15 seconds; 64° C., 30 seconds; 72° C., 1 minute. ForST90-375, the same conditions were used except that the annealingtemperature was 62.5° C. PCR products were analyzed on a 7% acrylamidegel.

Tumor Cell Lines and Chromosome Preparations

Lipoma specimens were obtained from patients at the time of surgery.Tumor culture, metaphase chromosome harvesting, slide preparation, andtrypsin-Giemsa banding were performed as described previously (Fletcheret al., 1991). Metaphases with rearrangements of chromosome 12 in bandq15 were identified and corresponding cell pellets stored in fixative at−20° C. were used to prepare slides for FISH. These slides were storedat room temperature for at least 10 days prior to hybridization.

Lambda clones shown in FIG. 1 were mapped to lipoma tumor metaphasechromosomes from ST90-375[46,XX,t(12;15)(q15;q24)], ST91-198[46,XX,t(12; 13)(q15;q21-32)], and ST93-724 [46,XX,t(3; 12)(q29;q15)]Karyotypes for lipomas ST90-375 and ST91-198 have been reportedpreviously (Fletcher et al., 1993).

FISH with Lambda Clones

Slides for FISH were prepared as recommended in the Hybridization Kit(Oncor, Gaithersburg, Md.) except for denaturation at 68° C. for 30seconds.

Lambda probes were labeled with digoxigenin-11-dUTP (BoehringerMannheim, Indianapolis, Ind.) using 1 mg of the appropriate lambda DNAusing dNTPs obtained from Boehringer Mannheim and the DNase I/DNApolymerase I mix from the BioNick Labeling System (BRL). Labelingreactions were performed at 16° C. for 2 hours. 500 ng ofdigoxigenin-labeled lambda probe was lyophilized with 5 mg of Cot-1 DNA(BRL) and resuspended in 20 ml deionized water. 2 ml of resuspendedprobe was added to 9 ml Hybrisol VI (Oncor). The lambda probe wasdenatured, hybridized to slides, and washed according to standardprotocols (Oncor). Digoxigenin-labeled lambda clones were detected usingthe fluorescein-labeled antidigoxigenin antibody (Oncor) according tothe manufacturer's recommendations. Metaphase chromosomes werecounterstained with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI)according to the protocol supplied by Oncor. Hybridization was observedusing a Zeiss Axioskop microscope and images captured with theCytoVision Imaging System (Applied Imaging).

FIGS. 1(A) and 1(B) illustrate the genomic structure of the human HMGI-Cgene. FIG. 1(A): 403, H409, H5003, H1001 and H4002 are genomic lambdaFIXII clones (see Materials and Methods) that contain the five exons(E1-E5) of the human HMGI-C gene. FIG. 1(B): Exons are denoted by boxesand introns by a line. Overlapping lambda clones were not obtainedwithin intron 3 and this region is denoted with a dashed line. Sequencesencoding potential functional domains, AUG and UAG codons are shown inthe exons. The A-T hook motifs of the DNA-binding domains are shown asstippled areas and the solid region (in E5) encodes for the acidicdomain of unknown function. The Figure is not drawn to scale because ofthe large 5′ and 3′ UTRs.

FIGS. 2(A) through 2(F) illustrate FISH mapping of HMGI-C lambda clonesto lipoma tumor metaphase chromosomes from three lipomas revealingrearrangement of HMGI-C in all three tumors. The normal chromosome 12homologs provide internal positive hybridization controls and are markedby yellow arrows in each metaphase, while derivative chromosomes aremarked by red arrows. Lambda clones H403 and H409 from the 5′ end ofHMGI-C were used as FISH probes to lipoma metaphase chromosomes fromFIG. 2(A) ST90-375 and FIG. 2(C) ST93-724, respectively. Notehybridization on the normal chromosome 12 and the der(12), demonstratingthat these clones map proximal to the breakpoint in both lipomas. Incontrast, when H403 was hybridized to lipoma metaphase chromosomes fromFIG. 2(E) ST91-198, hybridization was observed on the der(13) showing amap position distal to the breakpoint in this tumor. H4002 from the 3′end of HMGI-C was used as a FISH probe to lipoma metaphase chromosomesfrom FIG. 2(B) ST90-375 and FIG. 2(D) ST93-724; note hybridization onthe normal chromosome 12 and the der(15) or der(3), respectively,indicating that these clones map distal to the breakpoint in bothlipomas. However, FISH with H4002 from the 3′ end of HMGI-C on FIG. 2(F)ST91-198 revealed hybridization on the normal chromosome 12 only,suggesting this clone is deleted from either der (12) or der(15) in thistumor. Metaphase spreads were counterstained with DAPI. Lipomakaryotypes are: ST90-375, t(12;15)(q15;q24); ST93-724, t(3;12)(q29;q15);ST91-198, t(12;13)(q15;q21-32).

FIG. 3 illustrates RT-PCR amplification of HMGI-C chimeric transcripts.3′ RACE on RNA from lipomas ST90-375 (375) and ST93-724 (724) yield 441bp and 672 bp products. Reverse transcription was performed with anoligo-dT primer linked to an adapter sequence and was followed by anested PCR with sense primers from exon 1 and spanning exons 1 and 2.DLD-1 is a colorectal adenocarcinoma cell line that expresses wild-typeHMGI-C (data not shown) but under these conditions, the predicted 3.1 kbwild-type message was not amplified. Products were analyzed on a 1.5%agarose gel. M are molecular weight markers in kilobases.

FIG. 4 illustrates rearrangements of 12q15 in human lipomas whichdisrupt the HMGI-C gene and produce chimeric transcripts. HMGI-C denotesthe nucleotide and amino acid sequence of the wildtype gene and the openbox sequence corresponds to the end of HMGI-C exon 3. t(3;12) andt(12;15) refer to the nucleotide and predicted amino acid sequences ofthe chimeric transcripts from the cloned cDNA products obtained by 3′RACE on RNA isolated from primary cell cultures of ST93-724, t(3;12),and ST90-375, t(12;15), respectively. Chr. 3 and Chr. 15 refer to thenovel sequences derived from chromosome 3 or 15 in t(3;12) and t(12;15)lipomas, respectively. Only the sequences immediately adjacent to thefusion sites are shown.

FIG. 5 illustrates RT-PCR using primers located on either side of thefusion site between HMGI-C and novel sequences. RNA refers to the lipomasource of total RNA. Primer 375 is an oligonucleotide that iscomplementary to the novel sequence from the chimeric transcript oflipoma ST90-375 and is located 8 nucleotides downstream of the fusionpoint. Primer 724 is a complementary oligonucleotide to the novelsequence from the chimeric transcript of lipoma ST93-724 and is located425 nucleotides downstream of the fusion point. Total RNA from bothlipoma primary cell cultures was reverse transcribed using either 375 or724 primers and PCR amplified using HMGI-C sense primer (which spansexons 1 and 2) and the antisense primer used for reverse transcription.Expected product sizes are: 180 bp from ST90-375 cDNA with 375 primerand 597 bp from ST93-724 cDNA with 724 primer.

FIGS. 6(A) and 6(B) illustrate novel sequences fused to the DNAbinding-domains of HMGI-C which encode transcriptional regulatorydomains.

FIG. 6(A) illustrates a comparison of the novel chromosome 3 sequencefrom ST93-724 with the LIM domain-containing proteins, zyxin (Sadler etal., 1992), apterous (ap) (Cohen et al., 1992), Lh2 (Xu et al., 1993),Lin11 (Freyd et al., 1990), RBTN-1 (McGuire et al., 1989). Amino acidsthat constitute the LIM domain consensus are highlighted. The amino acidspacing between the consensus residues is indicated by an x. In additionto the totally conserved cysteine, histidine and aspartic acid residues(Sadler et al., 1992), LIM domains are characterized by the presence ofan aromatic residue adjacent to the first histidine and a leucinelocated C-terminal to the central HxxCxxCxxC cluster. The positions ofthese conserved residues are indicated by arrows. Each LIM domain isdesignated 1, 2 or 3 depending on its position relative to theN-terminus. The uninterrupted sequence of the two LIM domains in thevarious proteins are shown and gaps were introduced to permit alignmentof the two LIM domains.

FIG. 6(B) illustrates the potential transactivation acidic domainencoded by the sequence derived from chromosome 15 in ST90-375. Acidicresidues are underlined and the amino acids, serine and threonine, arein bold type.

FIG. 7 illustrates the structure and domain organization of HMGI-C andthe predicted fusion proteins. The vertical dashed line shows thelocation of junction sites in the chimeric products. DNA binding domainsof HMGI-C (AT) are preserved in the fusion proteins but the C-terminaldomain (stippled) is replaced by potential transcriptional regulatorydomains. LIM, LIM domain; (—), acidic domain; S,T, serine-threonine richdomain.

HMGI Proteins in Mammalian Growth and Development

FIGS. 8(A) through (D) illustrate the identification and genomiccharacterization of the HMGI-C gene at the pygmy locus in normal andmutant alleles. FIG. 8(A): Delineation of the overlapping deletedgenomic regions at the pygmy locus in the spontaneous and transgenicinsertional mouse mutants. The open box above clone 3 positions the 0.5kb ApaI—ApaI fragment and the filled boxes represent single copysequences used as probes to analyze genomic DNA isolated from mice ofvarying genotypes (Xiang, X. et al., 1990). Solid and dashed linesrepresent presence or absence of genomic sequences, respectively, in thetransgenic insertional mouse mutant pg^(TgN40ACha) (A) and thespontaneous mutant pygmy (pg). FIG. 8(B): Exon amplification from lambdaclones 803 and 5B. The primary PCR exon amplification products in bothsense (+) and antisense (−) orientations from the lambda clones shown inFIG. 8(A) were analyzed on a 5% polyacrylamide gel (Buckler, A. et al.,1991). The 379 bp PCR product observed in the control pSPL1 lane resultsfrom splicing between the HIV tat and b-globin vector sequences(Buckler, A. et al., 1991). FIG. 8(C): Sequence of exons amplified fromclone 803 and comparison to the HMGI-C gene. FIG. 8(D): A series ofoverlapping phage clones extending approximately 190 kb at the pygmylocus. The discontinuous region represents an unclonable 11 kb fragmentas estimated from Southern blots of cleaved genomic DNA probed withsingle copy sequences from the end of the clonable region. The positionand number of the HMGI-C exons (not drawn to scale) are shown above thewildtype locus. Single copy sequences were isolated at the indicatedpositions and are represented by filled boxes below the wildtype locus.Thick bars and blank regions represent the genomic sequences that arepresent or deleted in the two alleles.

Methods. The 0.5 kb ApaI—ApaI fragment (Xiang, X. et al., 1990) was usedas a probe to isolate clones 3 and 4 from an EMBL3 mouse genomic library(a kind gift of Dr. E. Lacy) and a YAC (902CO711) from a mouse YAClibrary (Lehrach, H. et al., 1990). YAC 902CO711 was further subclonedinto lambda FIX II (Ausubel, F. et al., 1988) and 86 clones thathybridized to radioactively-labeled mouse genomic DNA were picked andtransferred to new plates in a gridded array (Ausubel, F. et al., 1988).Lambda clones 802, 906, 5B, 803 and 308 were isolated after the walk wasinitiated with the 0.5 kb ApaI—ApaI fragment and accomplished byrepeated hybridization to filters of the array. Overlaps between thecontig clones and colinearity with the genome were confirmed by acombination of clone to clone and clone to genomic hybridizations alongwith restriction mapping. Exon amplification was performed (ExonTrapping System, Gibco BRL) after the genomic inserts from the lambdaclones were removed by cleavage with SalI, partially filled-in (Ausubel,F. et al., 1988) and subcloned into a partially filled-in BaniHl cleavedpSPL1 plasmid (Buckler, A. et al., 1991). The DNA was electroporatedinto COS-7 cells at 180V and 960mF in a Bio-Rad Gene Pulser. CytoplasmicRNA was isolated after 2-3 days and RT-PCR performed using primerssupplied by the manufacturer. The secondary PCR amplification products(Buckler, A. et al., 1991) from clones 803 and 5B were subcloned intothe plasmid vector, pAMP10 (Exon Trapping System, Gibco BRL) andsequenced using the Sequenase Version 2.0 sequencing kit (USB) (Ausubel,F. et al., 1988). A 344 bp fragment corresponding to the complete openreading frame of the HMGI-C gene (Manfioletti, G. et al., 1991) wasamplified from 12.5 dpc mouse embryos (see text) using reversetranscription (RT) and PCR. Lambda clones containing the HMGI-C exonswere then isolated by hybridization of the 344 bp radioactively-labeledfragment to the gridded array of lambda clones and subsequentlyconnected through chromosome walking. The RT-PCR conditions forisolation of the 344 bp fragment consisted of first strand cDNAsynthesis with primer 1 (5′-ATGAATTCCTAATCCTCCTCTGC-3′SEQ ID NO: 14),followed by PCR amplification with primers 1 and 2(5′-ATGGATCCATGAGCGCACGCGGT-3′SEQ ID NO: 14). PCR conditions were 94°C., 0.5 minute; 55° C., 0.5 minute; 72° C., 1 minute; for 30 cycles. Theamplified product was confirmed by sequencing analysis (Ausubel, F.etal., 1988).

FIG. 9 illustrates HMGI-C gene expression of three alleles at the mousepygmy locus. The wildtype allele is represented by +, the transgenicallele pg^(TgN40ACha) by A, the spontaneous mutant allele by pg and anallele at the pygmy locus which involves a paracentric inversion onchromosome 10 (In(10)17Rk) by Rk.

Methods. The genotypes were established for mice in line A and thespontaneous mutant pg as previously described (Xiang, X. et al., 1990),while mice containing the In(10)17Rk inversion were detected by aPCR-based RFLP (unpublished results). RNA was isolated from 12.5 dpcembryos and equal amounts (5 mg) were analyzed by Northern blothybridization (Ausubel, F. et al., 1988). The probes were a 138 bpnucleotide cDNA fragment encompassing exons 2 and 3 of the HMGI-C geneand a 340 bp cDNA fragment containing the complete coding sequence ofthe HMGI(Y) gene (Johnson, K. et al, 1988). The blot was subsequentlyhybridized to an oligonucleotide complementary to murine 28S ribosomalRNA (Barbu, V. & Dautry, F., 1989) to ensure equal amounts of RNA werepresent in each lane and the results are shown in the lower panel.

FIGS. 10(A) through(C) illustrate targeted disruption of the HMGI-Cgene. FIG. 10(A): Targeting strategy. Endogenous HMGI-C gene (top),targeting vector (middle) and predicted mutant gene (bottom). Thetargeting vector was created by replacing the 3 kb DNA fragmentcontaining exonl (E1) and exon2 (E2) with a PGK-neo cassette. The vectoralso includes a MC1-tk cassette at the 5′ end of the long homologoussegment. B, BamHI; Probe, a 4 kb HincII fragment used to identify thedisrupted allele. FIG. 10(B): Southern blot analysis of mice from aheterozygous cross. DNA from tails of the mice was digested with BamHIand hybridized to the external probe (see FIG. 10(A)). The positions ofthe bands corresponding to the wildtype allele (10.5 kb) and the mutantallele (9.3 kb) are indicated. FIG. 10(C): Western blot analysis ofwildtype (+/+), heterozygous (+/−) and homozygous (−/−) 12.5 dpc embryoswith anti-GST-HMGIC rabbit IgG.

Methods. Genomic clones of the mouse HMGI-C gene were isolated from themouse pygmy locus as described in FIG. 8 legend. Linearized vector (10mg) was electroporated into AB1 ES cells at 280V, 500 mF, and homologousrecombination events enriched for by selection with G418 (350 mg/ml) and2 mM gangcyclovir (Syntex) on SNL76/7 feeder cells. Six targeted cloneswere obtained and three were injected into C57BL/6J blastocysts togenerate chimaeras. Chimaeric males were mated to C57BL/6J females, andheterozygous offspring intercrossed to produce subsequent generations.Southern blot analysis of the progeny from heterozygous crosses wasperformed as described (Ausubel, F. et al., 1988) Proteins wereextracted from 12.5 dpc mouse embryos from a heterozygous cross withlysis buffer containing 50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mMmagnesium acetate, 0.2 mM EDTA, 1.0 mM PMSF, and 1% SDS. 10 mg of eachsample was separated by 15% SDS-PAGE, transferred to a nylon membrane(Duralon, Stratagene) and HMGI-C was detected using rabbit IgG antimouseGST-HMGI-C, HRP-conjugated goat anti-rabbit IgG and ECL substrate(Amersham).

FIGS. 11(A) through (C) illustrate expression of HMGI-C in developmentand growth. FIG. 11(A): Temporal expression pattern of HMGI-C andHMGI(Y) determined by Northern blot analysis of RNA (5 mg) isolated fromthe head (H) and body (B) of mouse embryos whose ages in days postcoitum are indicated at the top of the panel. No expression of HMGI-Cwas detected in placenta at any of these stages (data not shown). Theprobes are described in the legend of FIG. 9. FIG. 11(B): Spatiallocalization of HMGI-C transcripts in 11.5 dpc mouse embryos.Photomicrographs of 8 mm, adjacent, parasaggital sections through 11.5dpc mouse embryos hybridized with the antisense (A) or sense (B) strandof exon 2 and 3 of HMGI-C or stained histochemically with haematoxylinand eosin (C). G, gut mesenchyme; H, heart; L, liver; Lb, limb bud; M,mandible; N, median nasal process; NE, neural epithelium; 0, otocyst.Magnification: 25X. FIG. 11(C): Growth of wildtype and pygmy embryonicfibroblasts. Fibroblasts derived from 13.5 dpc embryos were seeded at aconcentration of 1.7×10³ cells per cm2 in DMEM containing 10% fetalbovine serum. Cell number (ordinate) was determined on day 4. Small barsrepresent standard deviations of triplicate experiments. P<0.001. Thegenotypes of embryos were determined as previously described (Xiang, X.et al, 1990)

Methods. For in situ hybridization, CBA/J embryos (11.5 dpc) were fixedin 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffinsections were deparaffinized and hybridized with sense and antisenseriboprobes corresponding to exons 2 and 3 of HMGI-C as previouslydescribed (Duncan, M. et al., 1992). Sections were stained withhaematoxylin and eosin according to standard procedures.

Translocation Breakpoints Upstream of the HMGI-C Gene in UterineLeiomyomata

Fluorescence in situ Hybridization (FISH)

Slides for FISH were prepared as recommended in the Hybridization Kit(Oncor, Gaithersburg, Md.), except for denaturation at 68° C. for 30seconds.

HMGI-C clones were in the lambda FIXII vector (Stratagene, La Jolla,Calif.). They were labeled with digoxigenin-1-dUTP (Boehringer Mannheim,Indianapolis, Ind.) with 1 μg of the appropriate lambda DNA, dNTPs fromBoehringer Mannheim, and the DNasel/DNA polymerase mix from the BioNickLabeling System (BRL, Gaithersburg, Md.). Labeling reactions wereperformed at 16° C. for 2 hours. Five hundred nanograms ofdigoxigenin-labeled probe were lyophilized with 5 μg of Cot-1 DNA (BRL)and resuspended in 20 μl of deionized water. Two microliters ofresuspended probe were added to 9 μl Hybrisol VI (Oncor). The probe wasdenatured, hybridized to slides, and washed according to standardprotocols (Oncor). Digoxigenin-labeled lambda-clones were detected withfluorescein-labeled antidigoxigenin antibody (Oncor) according to themanufacturer's recommendations, and metaphase chromosomes werecounterstained with 4,6diamidino-2-phenylindole-dihydrochloride (DAPI).Hybridization was observed with a Zeiss Axioskop microscope, and imageswere captured with the CytoVision Imaging System (Applied Imaging,Pittsburgh, Pa.)

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

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While the invention has been particularly described in terms of specificembodiments, those skilled in the art will understand in view of thepresent disclosure that numerous variations and modifications upon theinvention are now enabled, which variations and modifications are not tobe regarded as a departure from the spirit and scope of the invention.Accordingly, the invention is to be broadly construed and limited onlyby the scope and spirit of the following claims.

14 23 base pairs nucleic acid unknown unknown DNA (genomic) NO unknown 1AGGGGACAAC AAATGCCCAC AGG 23 25 base pairs nucleic acid unknown unknownDNA (genomic) NO unknown 2 CGTCACCAGG GACAGTTTCA CTTGG 25 32 base pairsnucleic acid unknown unknown DNA (genomic) NO unknown 3 GCAATACGACTCACTATAGT TTTTTTTTTT TT 32 19 base pairs nucleic acid unknown unknownDNA (genomic) NO unknown 4 CTTCAGCCCA GGGACAACC 19 19 base pairs nucleicacid unknown unknown DNA (genomic) NO unknown 5 GCAATACGAC TCACTATAG 1919 base pairs nucleic acid unknown unknown DNA (genomic) NO unknown 6GGAAGCAGCA GCAAGAACC 19 20 base pairs nucleic acid unknown unknown DNA(genomic) NO unknown 7 CTTCTTTCTC TGCCGCATCG 20 20 base pairs nucleicacid unknown unknown DNA (genomic) NO unknown 8 GTGAGGATGA TAGGCCTTCC 2020 base pairs nucleic acid unknown unknown DNA (genomic) NO unknown 9CAGAAGCAGA CCAGCAAACC 20 20 base pairs nucleic acid unknown unknown DNA(genomic) NO unknown 10 CTTCTTTCTC TGCCGCATCG 20 20 base pairs nucleicacid unknown unknown DNA (genomic) NO unknown 11 CTCTGGAGCA GTGCAATGTG20 20 base pairs nucleic acid unknown unknown DNA (genomic) NO unknown12 GTGAGGATGA TAGGCCTTCC 20 23 base pairs nucleic acid unknown unknownDNA (genomic) NO unknown 13 ATGAATTCCT AATCCTCCTC TGC 23 23 base pairsnucleic acid unknown unknown DNA (genomic) NO unknown 14 ATGGATCCATGAGCGCACGC GGT 23

We claim:
 1. A method for detecting high mobility group DNA-bindingproteins (HMGI-C) as a diagnostic marker for benign mesenchymal orlipoma rumors using a probe for a sample, or an extract of the sample,from a patient that recognizes HMGI-C, which comprises the steps of: (a)contacting HMGI-C from a sample from a patient with a probe which bindsto HMGI-C; and (b) analyzing for HMGI-C by detecting levels of the probebound to the HMGI-C; (c) treating a control sample according to themethod to assess the level of HMGI-C in the control sample; wherein thepresence of HMGI-C in the sample in step (b) in an amount higher thanthe amount in the control sample in step (c) is diagnostic for a benignmesenchymal or lipoma tumor.
 2. The method according to claim 1, whereinthe sanple is selected from the group consisting of a biopsy sample, aurine sample, a blood sample, a feces sample, and a saliva sample. 3.The method according to claim 1, wherein the method is selected from thegroup consisting of a histological assay, biochemical assay, flowcytometry assay, Western blot assay, and capture assay.
 4. A methdod fordetecting high mobility group DNA-binding proteins (HMGI-Y) as adiagnostic marker for a benign mesenchymal tumor using a probe for asample, or an extract of the sample, from a patient that recognizesHMGI-Y, which comprises the steps of: (a) contacting HMGI-Y from asample from a patient with a probe which binds to HMGI-Y; and (b)analyzing for HMGI-Y by detecting levels of the probe bound to theHMGI-Y; (c) treating a control sample according to the method to assessthe level of HMGI-Y in the control sample; wherein the presence ofHMGI-Y in the sample in step (b) in an amount higher than the amount inthe control sample in step (c) is diagnostic for a benign mesenchymaltumor.
 5. The method according to claim 4, wherein the sample isselected from the group consisting of a biopsy sample, a urine sample, ablood sample, a feces sample, and a saliva sample.
 6. The methodaccording to claim 4, wherein the method is selected from the groupconsisting of a histological assay, biochemical assay, flow cytometryassay, Western blot assay, and capture assay.
 7. A method for detectinghigh mobility group DNA-binding proteins (HMGI-C or HMGI(Y)) as adiagnostic marker for malignant tumors using a probe for a sample, or anextract of the sample, from a patient that recognizes HMGI-C or HMGI(Y),which comprises the steps of: (a) contacting HMGI-C or HMGI(Y) from asample from a patient with a probe which binds to HMGI-C or HMGI(Y),respectively; and (b) analyzing for HMGI-C or HMGI(Y) by detectinglevels of the probe bound to the HMGI-C or HMGI(Y), respectively; (c)treating a control sample according to the method to assess the level ofHMGI-C or HMGI(Y) in the control sample; wherein the presence of HMGI-Cor HMGI(Y) in the sample in step (b) in an amount higher than the amountin the control sample in step (c) is diagnostic for a malignant tumor.8. The method according to claim 7, wherein the sample is selected fromthe group consisting of a biopsy sample, a urine sample, a blood sample,a feces sample, and a saliva sample.
 9. The method according to claim 7,wherein the method is selected from the group consisting of ahistological assay, biochemical assay, flow cytometry assay, Westernblot assay, and capture assay.